We have identified several growth factors and cytokines that can protect neurons against dysfunction and death in experimental models of Alzheimer?s disease, Parkinson?s disease and stroke. These trophic factors activate signaling pathways that stimulate the expression of genes whose encoded proteins increase resistance of neurons to oxidative and metabolic stress. We are also identifying novel intracellular proteins that promote neuronal survival and plasticity, including kinases and telomerase. We have established a role for telomerase in regulating neuronal survival during brain development, and have obtained evidence that turning on telomerase in neurons in the adult brain can protect neurons in experimental models of neurodegenerative disorders. Finally, our studies of neural stem cells are revealing novel approaches for replacing lost neurons in animal models of neurodegenerative disorders. --Neuroprotective Signaling Via Integrins and Pathways Involving Akt and NF-kappaB: Integrins are integral membrane proteins that mediate adhesive interactions of cells with the extracellular matrix and with other cells. Integrin engagement results in activation of intracellular signaling cascades that effect several different cellular responses including motility, proliferation and survival. Although integrins are known to provide cell survival signaling in various types of non-neuronal cells, the possibility that integrins modulate neuron survival has not been explored. We have obtained data demonstrating a neuroprotective function of integrins in embryonic hippocampal neurons. Neurons grown on laminin, an integrin ligand, exhibit increased resistance to glutamate-induced apoptosis compared with neurons grown on polylysine. Neurons expressed integrin beta1 and treatment of cultures with an antibody against integrin beta1 abolished the protective effect of laminin. Neurons maintained on laminin exhibited a sustained activation of the Akt signaling pathway demonstrated in immunoblot analyses using an antibody that selectively recognizes phosphorylated Akt. The neuroprotective effect of integrin engagement by laminin was mimicked by an IKLLI-containing integrin-binding peptide and was abolished by treatment of neurons with the PI3 kinase inhibitor wortmanin. Levels of the anti-apoptotic protein Bcl-2 were increased in neurons grown on laminin and decreased by wortmanin, suggesting a mechanism for the neuroprotective effect of integrin-mediated signaling. The ability of integrin-mediated signaling to prevent glutamate-induced apoptosis suggests a mechanism whereby neuron-substrate interactions can promote neuron survival under conditions of glutamate receptor overactivation. Prototypical NF-kappaB consists of a transcription factor dimer of p50 and p65, and an inhibitory subunit called I-kappaB. NF-kappaB is activated in neurons in response to excitotoxic, metabolic, and oxidative stress. Cell-culture data suggest that activation of NF-kappaB can prevent neuronal apoptosis, but its role in vivo is unclear and the specific kappaB subunits involved are unknown. In Huntington's disease (HD), striatal neurons degenerate, and a similar pattern of neuronal vulnerability occurs in rats and mice following exposure to the mitochondrial toxin 3-nitropropionic acid (3NP). We report that mice lacking the p50 subunit of NF-kappaB exhibit increased damage to striatal neurons following administration of 3NP. The neuronal death occurs by apoptosis as indicated by increased caspase activation and DNA fragmentation into oligonucleosomes. NF-kappaB activity is markedly increased in striatum 24-72 h following 3NP administration in wild-type mice, but not in mice lacking p50, indicating that p50 is necessary for the vast majority of 3NP-induced NF-kappaB DNA-binding activity in striatum. Cultured striatal neurons from p50-/- mice exhibited enhanced oxidative stress, perturbed calcium regulation, and increased cell death following exposure to 3NP, suggesting a direct adverse effect of p50 deficiency in striatal neurons. --Neuroprotective Actions of Telomerase: Telomerase is an enzyme activity consisting of a reverse transcriptase called TERT and an RNA component that adds repeats of a DNA sequence (TTAGGG) to the ends of chromosomes, thereby preventing their shortening. We have found that telomerase activity is high in the brain at embryonic day 13 (E13), declines markedly between E13 and E18, remains at a low level until postnatal day 3 (P3) and becomes undetectable by P10. Surprisingly, the temporal pattern of change in telomerase activity is not paralleled by a decrease in levels of TERT mRNA that remain elevated from E13 to P5 (with fluctuations during this time window that vary among brain regions), and then decrease to a lower level that is maintained into adulthood. TRF1 and TRF2 mRNA levels are relatively constant throughout brain development. Our data are consistent with a role for telomerase activity in proliferation of neural progenitor cells, and further suggest that TERT may play roles in neuronal differentiation and survival. The dissociation between TERT expression and telomerase activity is a novel finding that suggests biological functions for TERT in addition to telomere maintenance. In additional studies we found that TERT can protect cultured neural cells against apoptosis induced by DNA-damaging chemotherapeutic drugs. These findings suggest that telomerase may suppress DNA damage or an apoptotic signal activated by DNA damage. Overexpression of wild-type human TERT in HeLa cells, and in a cells lacking TERT but containing the telomerase RNA template, suppresses cytochrome c release and caspase activation and increases their resistance to apoptosis induced by the DNA damaging agent etoposide or the bacterial alkaloid staurosporine. In contrast, TERT mutants with disruptions of either the RT domain or a 14-3-3 binding domain required for nuclear localization fail to protect cells against apoptosis, and overexpression of TERT in cells lacking the telomerase RNA template is also ineffective in preventing apoptosis. Additional findings show that TERT suppresses apoptosis at an early step prior to release of cytochrome c from mitochondria and prior to nuclear translocation of apoptosis-inducing factor. We conclude that both nuclear localization and RT activity are required for the anti-apoptotic function of TERT in tumor cells, and that TERT can suppress a nuclear signal(s) that is an essential component of apoptotic cascades triggered by diverse stimuli. We have also been working to identify signaling pathways that regulate TERT expression. We screened numerous neurotrophic factors and cytokines to determine whether they affected telomerase activity in cultured dissociated embryonic rat hippocampal neurons. BDNF and sAPPalpha induced increases in telomerase activity, and increases in levels of TERT mRNA and protein, whereas bFGF, NGF and IGF-1 had no effect. We investigated the signal transduction pathway(s) by which BDNF and sAPPalpha increase telomerase levels and found that Akt kinase and mitogen-activated protein (MAP) kinases played important roles. We have recently produced multiple transgenic lines of mice overexpressing TERT in the nervous system. This was accomplished using a plasmid in which the expression of TERT is driven by a prion protein promoter. We have chosen four lines of mice that overexpress TERT, at varying amounts, in the brain and spinal cord, and are in the process of examining the mice to determine whether they exhibit abnormalities. The TERT transgenic mice survive and reproduce well. Thus far we have observed an abnormality in eye development in all 4 lines of TERT transgenic mice, and we are currently in the process of determining whether other abnormalities are also present.